We analyzed clinical factors and graft survival associated with complement-dependent microcytotoxicity (CDC) crossmatch (XM) positive (+) kidney transplants in 1995 to 2009 United Network of Sharing (UNOS) registry data. CDCXM negative (-) transplants were selected from centers and years in which at least one CDCXM+ transplant was performed at a given center in a given year. CDCXM+ and CDCXM- results were compared with bivariate and multivariate survival analysis. Our observations are as follows: (1) The risk of graft loss with CDCXM+ vs. CDCXM- results was markedly lower than the risk observed historically, e.g., living donor (LD)-CDCXM+ absolute all-cause graft survival reductions were 0.7% at 24 hours (P=0.007), 2.9% at one year (P <0.0001), 3.7% at five years (P<0.0001); deceased donor (DD)-CDCXM+ absolute graft survival reductions were 0.7% at 24 hours (P=0.02), 3.5% at one year (P <0.0001), 2.7% at five years (P=0.0009). On covariate adjustment, the only significant association of CDCXM+ vs. CDCXM- results was with one-year graft loss risk: LD aHR 1.44 (95% CI 1.05-1.96), DD aHR 1.33 (CI 1.10-1.61). (2) CDCXM+ transplantation was more commonly performed among groups disadvantaged with respect to transplant access, including sensitized, previously transplanted women and black recipients. (3) In CDCXM+ recipients, there was a high percentage of flow cytometry (FC) XM- and autoXM+ results. After removing these groups, outcomes with CDCXM+ results were relatively good. (4) CDCXM+/FCXM+ vs. CDCXM-/FCXM- graft loss risk was observed only in LD recipients transplanted at centers performing fewer than 10 such transplants during the study period: 11.0% reduction (P<0.0001) and aHR of 2.86 (CI 1.18-6.94) at one year; 14.7% reduction (P<0.0001) and aHR of 1.77 (CI 0.88-3.58) at five years. Although using CDCXM+ as a contraindication to transplantation has been associated with virtual elimination of hyperacute rejection, the negative effect of a CDCXM+ in contemporary practice is relatively small, questioning the value of the CDCXM as a standalone test.

The landmark study of Medawar demons­trated that naïve transplant recipients mobilize their immune systems for the purpose of re­jecting allotransplants, and that retransplanted recipients often do so in an accelerated fashion as an indication of sensitization. [1] In 1969, Patel and Terasaki reported a strong association bet­ween the presence of a positive complement-dependent microcytotoxicity crossmatch (CDCXM+) and immediate kidney graft loss. [2] As an outgrowth of that report, the presence of CDCXM+ generally has been considered as a contraindication to kidney transplantation.

Much has changed in transplantation since 1969, including changes in crossmatch [3],[4],[5],[6] and antibody screening techniques, [7],[8] which have been modified to increase specificity and sensitivity. Currently, maximal specificity and sensitivity are achieved by flow cytometry crossmatch (FCXM) and solid phase antibody-screening technology. Not infrequently, pa­tients who are FCXM positive (FCXM+) are transplanted, and less frequently, transplants proceed with CDCXM+ results. The rarity of transplantation with CDCXM+ results presents a challenge for understanding associated out­comes in single or even multicenter contexts.

We aimed this study at advancing the un­derstanding of CDCXM+ transplantation in contemporary practice; therefore, we analyzed national registry transplant data from 1995 to 2009, considering the relationship between cli­nical factors, variations in XM technology and outcomes associated with performance of transplants with CDCXM+ results.

Methods

Data were drawn from United Network of Sharing (UNOS) Standard Transplant Analysis and Research files of the related Organ Pro­curement and Transplant Network (OPTN) describing kidney-alone transplants performed during 1995 to November 2009. [9] At the time of transplant, information was transmitted from each center to the OPTN on the cross-match technique, cellular target used, and the type of antibody detected for allo- and auto-crossmatch. We studied transplants with re­ported CDCXM data submitted from 30 days prior to transplant until the day of transplan­tation.

Crossmatch techniques and results

The UNOS database allows entry of three CDCXM techniques (NIH, wash, and anti-human globulin (AHG)). In previous analyses by others [10] and us (unpublished data) no con­sistent pattern of outcome differences between CDCXM techniques were found, so we did not distinguish patients according to CDCXM tech­nique in the current study. Although some laboratories report only T cell results, virtually no laboratories report only B cell results. Thus, outcome results are available for crossmatches using T and B targets or T only targets. Two CDCXM+ groups were studied: T-B+ and T+ which included T+B+ and T+B not done, missing or indeterminate. Although we exclu­ded T+B- results, the T+B not done, missing or indeterminate group may include a small fraction of T+B-. In most analyses we studied T+ and T-B+ separately. Infrequently, because of group size, we combined T+ and T-B+ data. We excluded T weak positive and in­determinate and B weak positive and indeter­minate results not associated with a clear-cut positive result with the other cell type.

We categorized CDCXM+ recipients accor­ding to the type of their antibody, IgG only vs. IgG and/or IgM and allo- vs. auto-antibody. Although data are available on IgG vs. IgG and/or IgM per se, we have chosen, because the standard FCXM uses an anti-IgG FITC label to identify anti-HLA antibody, to use FCXM+ to indicate IgG only, and, because both IgG and IgM fix complement, we have used CDCXM+ to indicate IgG and/or IgM.

Recipients of ABO incompatible transplants were excluded. Our CDCXM- group was li­mited to T-B- crossmatches. To reduce the impact of center or practice characteristics associated with the decision to perform CDCXM+ transplantation, CDCXM- trans­plants were selected from centers and years in which at least one CDCXM+ transplant was performed at a given center in a given year. The primary outcome of interest was all-cause allograft loss from graft failure or patient death. Graft failure is indicated by the permanent return of the transplant patient to dialysis or retransplantation according to OPTN reports. Death events were identified by OPTN reporting and supplemented with the Social Security Death Master File. Other variables collected in the OPTN registry at transplant are shown in [Table 1]. Panel reactive assay (PRA) data were not classified for HLA classes 1 and 2 in the registry prior to 2004 and were so considered as a total maximum percentage.

Table 1. Distributions of CDCXM results according to baseline clinical characteristics among deceased and living donor transplant recipients in the study sample.a

Statistical analyses were performed with SAS for windows software, version 9.2 (SAS Ins­titute, Inc., Cary, NC). Analyses were per­formed separately for recipients of deceased (DD) and living donor (LD) grafts. The dis­tributions of baseline clinical characteristics among patients transplanted with CDCXM+ vs. CDCXM- were compared by the chi-square test. We modeled associations of the likelihood of CDCXM+ vs. CDCXM- trans­plantation (adjusted odds ratio, aOR) by step-wise multivariable logistic regression, with P-value for retention of <0.05. Time-to-all-cause graft loss was censored at last expected UNOS follow-up or end of the study (November 2009). Variation in graft survival according to CDCXM results was compared by the Kaplan- Meier method, and the log-rank test was used to assess the statistical significance of differences in absolute survival. Survival was com­pared from transplant to the first and fifth anniversaries. Multivariate Cox regression was used to examine associations of CDCXM+ vs. CDCXM- with the relative risk of graft failure after adjustment for all other reported baseline clinical factors. To assess the potential impact of the frequency of crossmatch positive trans­plantation, which may be a surrogate marker for centers performing antibody reduction protocols, subanalyses were performed strati­fying the regression models by "volume" of CDCXM+/FCXM+ transplantation, where high and low volume were defined by performed of >=10 or <10 CDCXM+/FCXM+ transplants at the center during the study period.

Results

Among 10,261 eligible living donor (LD) transplants performed in the study period, the CDCXM technique was NIH in 2458, wash in 1125, and AHG in 4072. CDCXM+ results were present in 1044 (10.2%) of these transplants, of which 339 were CDCXM T+B+ or T+B not evaluable and 705 were CDCXM T-B+ [Table 1]. Of 15,438 eligible deceased donor (DD) transplants, the CDCXM tech­nique was NIH in 3796, wash in 2431, and AHG in 4194. CDCXM+ results were present in 1221 (7.9%), of which 396 were CDCXM T+B+ or T+B not evaluable, and 825 were CDCXM T-B+.

Clinical correlates of CDCXM+ transplantation Distributions of T-B-, T+ or T-B+ CDCXM results according to recipient, donor, and trans­plant characteristics are shown in [Table 1]. CDCXM+ transplantation was more common among younger compared to older patients, female compared to male recipients, sensitized patients, and retransplant recipients. For example, CDCXM T+ and CDCXM T-B+ trans­plants comprised 16.0% and 16.6%, respec­tively, of transplants performed among LD recipients with PRA>30%, compared to 1.9% and 5.5% of transplants among LD recipients with PRA 0-10%. By multivariable logistic regression, there was a strong and graded association of pre-transplant PRA level with the likelihood of transplantation with CDCXM+. Among LD recipients, T+CDCXM transplantation was more than 12 times as likely among recipients with PRA ≥81 vs. ≤10 (aOR 12.57, 95% CI 8.63- 18.31), and T-B+CDCXM+ transplantation was more than 4 times as likely among recipients with PRA ≥81 vs. ≤10 (aOR 4.67, 95% CI 3.32-6.57) [Table 2]. Similar patterns were observed among DD recipients. Trans­plantation with CDCXM+ also was more likely among retransplant compared to first trans­plant, female compared to male, and black vs. nonblack recipients, although associations were less strong than observed for PRA (aOR 1.18- 1.81).

Table 2. Variation in likelihood of CDCXM+ transplantation (T+ or T−B+) according to baseline clinical traits among transplants performed.

Associations of CDCXM+ with graft survival Kaplan-Meier graft survival estimates at 24 hours, one year, and five years after transplant according the crossmatch results among LD and DD recipients are shown in [Table 3]. LD-CDCXM+ recipients, compared to LD-CDCXM- recipients, had absolute graft sur­vival reductions of 0.7% at 24 hours (P = 0.007), 2.9% at one year (P <0.0001) and 3.7% at five years (P <0.0001). DD-CDCXM+recipients had absolute graft survival reduc­tions of 0.7 % at 24 hours (P=0.02), 3.5 % at one year (P <0.0001) and 2.7% at five years (P=0.0009). Compared to CDCXM- transplants, CDCXM T+ or T-B+ showed similar patterns of difference, with CDCXM T+ reductions being generally slightly higher than CDCXM T-B+ reductions. At five years post-trans­plant, LD-CDCXM T+ recipients, compared to LD-CDCXM- recipients, had an absolute graft survival reduction of 6.1 % (P<0.0001 ) and DD-CDCXM T+ recipients had an insigni­ficant absolute graft survival reduction of 2.4% (P=0.1).

Table 3. Graft survival at 24 hours, 1 year, and 5 years according to CDCXM, FCXM, and AutoXM status.

The role of IgG (as indicated by FCXM+) vs. IgG/IgM (as indicated by CDCXM+) in graft survival

Among 897 recipients of LD-CDCXM+ trans­plants, 357 also underwent FCXM, 201 of which were FCXM- and 156 FCXM+. LD-CDCXM+/FCXM+ vs. LD-FCXM/CDCXM- recipients showed 6.4% (P <0.0001) and 5.3 % (P=0.0004) one and five year graft survival reductions [Table 3]. Stratification of CDCXM+ into CDCXM T+ and T-B+ demonstrated si­milar graft survival compared to all CDCXM+ transplants. Notably, compared to LD-CDCXM+/ FCXM-, LD-CDCXM+/FCXM+ recipients also showed 7.1% one-year (P=.004) and 7.5% five-year (P=.006) graft survival reductions. Once again, stratification of the CDCXM according to T+ and T-B+ results showed similar outcomes. In contrast, no significant differences were seen between LD-CDCXM+/ FCXM- and CDCXM-/FCXM- graft survival at one or five years, even when the CDCXM was stratified for T+ and T-B+. Among 968 recipients of DD-CDCXM+ transplants, 259 also underwent FCXM, of which 122 were FCXM- and 137 FCXM+. In general, similar patterns in the various comparisons were seen, but levels of significance generally were not reached [Table 3]. One unexpected result was noted. The 49 CDCXM T+/FCXM+ recipients had notably good graft survival at one (85.7%) and five years (83.7%).

The role of autoantibody in graft survival

Among recipients of 897 LD-CDCXM+ trans­plants, 299 were reported as being autoXM-and 229 as being autoXM+. Among the autoXM+ recipients, there was no difference between the CDCXM+ vs. CDCXM- graft survival at one or five years, even when the CDCXM was stratified according to T+ and T-B+ results [Table 3]. In the autoXM- recipients, graft survival among the CDCXM+ recipients was significantly worse than among the CDCXM- outcomes at five years only (4% reduction, P=0.0003). Although the reduction persisted with T+ and T-B+ stratification, sig­nificance persisted only for T-B+ results. Among recipients of 968 DD-CDCXM+ trans­plants, 177 were reported as autoXM- and 180 autoXM+. Relative differences in graft survival according to CDCXM results were similar to patterns observed among LD recipients but of a lesser magnitude. Relative differences in graft survival accor­ding to CDCXM results by multivariable Cox regression are shown in [Table 4]. After covariate adjustment among LD recipients, CDCXM+ compared to CDCXM- transplantation was associated with 44% (aHR 1.44, 95% CI 1.05- 1.96) increase in the relative risk of one-year graft loss, and a 24% increase in the relative risk of five-year graft loss. Stratification of the CDCXM according to T+ and T-B+ revealed that significance was limited to CDCXM T+ transplants. Specifically, compared to CDCXM- transplants, CDCXM T+ results were asso­ciated with 71% (aHR 1.71, 95% CI 1.10- 2.68) and 50 % (aHR 1.50, 95% CI 1.10-2.05) increases in the adjusted relative risks of graft loss and one and five years, respectively. CDCXM T+/FCXM+ transplantation was associated with more than 2.5 times the relative risk of one-year graft loss compared to CDCXM- /FCXM- transplantation (aHR 2.69, 95% CI 1.19-6.08). FCXM results were indepen­dently prognostic, as CDCXM T+/FCXM+ transplantation was associated with 2-3 times the relative risk of one and five year graft loss compared to CDCXM+/FCXM- results. Among DD recipients, transplantation was associated with an increase in the adjusted relative risk of graft loss only for CDCXM T-B+ at one year compared to CDCXM T-B-(aHR 1.26, 95% CI 1.00-1.62).

Variation in outcomes according to center volume of CDCXM+/FCXM+ transplantation

To assess if the volume of crossmatch-positive transplants performed per transplant center is associated with outcome, we stratified results according to the performance of ≥10 "high volume" or <10 "low volume" CDCM+/ FCXM+ transplants during the study period. A significant reduction in graft survival was seen only in LD CDCXM+/FCXM+ vs. LD CDCXM-/ FCXM- transplants in centers that performed <10 CDCXM+/FCXM+ transplants (0-1 year: 11% reduction, P<0.0001, aHR 2.86, 95% CI 1.18-6.94; 0-5 years: 4.4% reduction, P<0.0001, aHR 1.77, 95%CI .88- 3.58). No significant reduction in 5-year graft survival was seen in DD CDCXM+/FCXM+ in centers that performed <10 CDCXM+/FCXM+ transplants or in LD or DD CDCXM+/FCXM+ transplants performed in centers that performed >10 CDCXM+/FCXM+ transplants during the study period [Table 5].

Since Patel and Terasaki's landmark study reporting the dire consequences of kidney transplantation in the presence of CDCXM+, CDCXM+ has been considered to be a contra­indication to transplantation. Nevertheless, some transplants do proceed despite CDCXM+. In the current study, we examined national registry data for a cohort of recent transplant recipients to define clinical correlates and out­comes associated with CDCXM+ among transplants. Our results included several key observations: (1) Among transplants performed, the risk of graft loss with CDCXM+ vs. CDCXM- results was markedly lower than the risk observed historically, particularly immediate graft loss. (2) CDCXM+ transplantation was more commonly performed among groups disadvantaged with respect to transplant access and/or outcome, i.e., high PRA, prior transplants, women, and black recipients. (3) In CDCXM+ recipients, there were a high percentage of FCXM- and autoantibody+ recipients reported. After removing these groups, outcomes of patients transplanted with CDCXM+ results were relatively good. (4) After covariate adjustment, the graft loss risk associated with CDCXM+/FCXM+ transplantation was limited to LD recipients at centers with a low volume practice in such transplants. The 1969 Patel-Terasaki study described a 77.4% reduction in graft survival in the im­mediate postoperative period associated with a CDCXM+ result. [2] In Gebel, Bray, and Nickerson's 2003 review of 23 reports, the median one-year graft survival reduction asso­ciated with CDCXM and/or FCXM+ results was 12% among first transplant recipients and 35% among retransplant recipients. [11] In a recent study, we reported FCXM+ to be asso­ciated with a 1.8-6.2% reduction in one year graft survival depending on the type of donor and the lymphocyte target used. [12] In the current study, one year graft loss reduction in recipients with CDCXM+ vs. CDCXM- was 2.4% and 3.8% in LD and DD recipients. Adjusting for confounding factors, including induction and maintenance immunosuppression regimens, further reduced the significance of CDCXM+ results .

To what factors can this reduction in graft loss improved outcomes in transplanted CDCXM+ recipients be attributed? Certainly in the current study and probably in the Patel- Terasaki study, transplants performed after CDCXM+ results reflect a small minority of all potential CDCXM+ recipients. In com­paring our results with those of Patel and Terasaki, an important consideration is the evolution of selection factors in the decision to proceed with transplantation in CDCXM+. In the Patel-Terasaki report, 92% were first transplants, 62% were male, and 65% were LD recipients, a distribution associated with rela­tively good outcome. In the current study, pa­tients with most recent PRA>50%, retransplant, female, and black recipients were signi­ficantly overrepresented among CDCXM+, groups associated with poor outcome. Thus patient demographics alone do not appear to explain improved outcome in the current era. It should be noted that these traits define groups with reduced access to transplantation and sug­gest that centers are willing to accept inferior outcomes in order to expand transplant access to disadvantaged patients.

The greatest change in outcome since 1969 has been the virtual elimination of immediate graft loss in CDCXM+ recipients. The Patel- Terasaki study noted immediate graft loss in 80% of CDCXM+ and 2.6% recipients of CDCXM-/PRA- recipients, whereas, in the current analysis, 24-hour graft loss occurred in 1.3% of LD and 1.6 % of DD CDCXM+ recipients. Although improved surgical and preservation techniques certainly have played a role in reduction of immediate graft loss since 1969, they cannot explain the difference in graft loss among CDCXM+ and CDCXM- in the 1969 study and the improvement in CDCXM+ vs. CDCXM- outcomes since then. Although there was no presentation of patho­logical information, it has been assumed that the major cause of immediate graft loss in those transplanted with CDCXM+ in the Patel-Terasaki series was hyperacute rejection. A possible explanatory factor for the markedly lower risk of hyperacute rejection after CDCXM+ transplants performed in modern practice may be selection of recipients with low anti-HLA titers. The relatively insensitive CDCXM technique of 1969 undoubtedly required the presence of high titers of antibody to show a positive reaction in contrast to today's CDCXM technique, which has under­gone multiple modifications to increase sensitivity. Titer data are not available in either Patel-Terasaki's data or current OPTN records.

Because of these findings of a relatively small effect of CDCXM+ results on graft loss risk among transplants performed, it is important to consider potential flaws or omissions in the analyses, such as entry or lab error. OPTN data are deidentified and it is not possible to trace individual patients and assure that there has not been an entry error. By their nature, such errors would be random and one would not expect to see association patterns such as differential likelihood of CDCXM+ results among sensitized patients as were ob­served in this analysis.

A surprising 55% of CDCXM+ transplants were FCXM-. This can be explained by a false positive CDCXM, a false negative FCXM, or by IgM as the responsible antibody. The ob­servation that CDCXM+/FCXM+ outcomes are significantly worse than CDCXM+/FCXM-among LD recipients and a similar trend exists among DD recipients makes false negative FCXM unlikely. The similarity of outcome of CDCXM+/FCXM- and CDCXM-/FCXM- results allows the possibility of a false positive CDCXM result and could explain the good outcomes in this group. A CDCXM+/FCXM- result also could be explained by the presence of only IgM antibody, antibody that would not be detected by the usual anti IgG-FITC label that is used in the standard FCXM, but would be detected by the standard CDCXM, which detects IgG and/or IgM antibody. The presence of donor-specific IgM and the absence of IgG antibody could explain the relatively good outcomes noted in the CDCXM+/ FCXM- group. By excluding recipients with CDCXM+/ FCXM- results, the possibility of including CDCXM false positive and IgM antibody is minimized. Even after these exclusions reci­pients with CDCXM+ results show relatively modest reductions in absolute graft survival. Of those transplanted with CDCXM+ results, a high percentage were reported as also having autoantibody. When the CDCXM+ recipients were stratified into autoXM+ and autoXM- groups, CDCXM+ results were detrimental only in LD-autoXM- recipients at five years. Although some of the patients transplanted with a CDCXM+ result might have been in­advertent, others may have been transplanted purposefully, considering the risk of transplan­ting with a CDCXM+ result to be less than not transplanting and the likelihood of finding a CDCXM- donor to be small. Of those trans­planted purposefully, some may have been untreated and some may have been trans­planted following antibody reduction therapy that did not remove all antibody. Available OPTN data do not contain information on use of antibody reduction treatments such as IVIG therapy and/or plasmapheresis; therefore we are not able to differentiate these two groups. Good outcomes have been reported with this latter group. [13],[14] Much has been reported on the immunomodulatory effects of IVIG be­yond simple antibody removal and plasmapheresis likely exerts an immunomodulatory effect as a result of cytokine removal explaining such good outcomes. [15]

In an attempt to determine if a center effect played a role in positive crossmatch outcome, we stratified for centers that performed ≥10 or <10 CDCXM+ transplants during the study period. We excluded CDCXM+/FCXM- transplants to avoid CDCXM+ results associated with false positive or IgM antibody. Interes­tingly, CDCXM+/FCXM+ results had the greatest negative effect for LD recipients transplanted at centers that performed <10 CDCXM+ transplants during the study period, with 2.9-times the adjusted relative risk of graft loss compared to CDCXM-/FCXM- at 1 year compared to an insignificant 1.22 times increase in centers performing >10 CDCXM+ transplants. By contrast, outcomes with CDCXM+/FCXM+ results did not vary by center practice in DD recipients, among whom CDCXM+/FCXM+ was not an adverse predictor after covariate adjustment. Although antibody reduction information is not available to us, centers doing <10 CDCXM+ transplants during the study period are unlikely to have antibody reduction protocols.

In conclusion, although consideration of the CDCXM+ result as a contraindication to transplantation has been the associated with the virtual elimination of hyperacute rejection, the negative effect of a CDCXM+ result in contemporary practice is relatively small com­pared to other accepted confounding clinical factors such as a second transplant or an expanded criteria donor. Current approaches to recipient evaluation focus on the outcome effect of the presence of donor-specific anti­body in the absence of a positive CDCXM, [16] but not on the outcome effect of a CDCXM+ in the presence or absence of donor-specific antibodies. Even in the absence of such anti­bodies, a positive CDCXM usually results in the elimination of such a potential recipient from consideration for transplant and stops subsequent testing.

The results of the current study question the value of the standard CDCXM as a stand­alone test. The prognostic value of a con­comitant FCXM is apparent from the data presented although neither the CDCXM nor FCXM distinguishes between HLA and non-HLA antibodies. As urged by Eckels, [17] we must be prepared for change. While these data should not be taken to justify eliminating the CDCXM, further work to determine under what circumstances CDCXM+ transplants can prospectively precede with safety is warranted.

Acknowledgements

An abstract describing portions of this work was presented at the 2011 American Transplant Congress on May 1, 2011, Philadelphia, PA.

Dr. Lentine received support from a grant from the National Institute of Diabetes Digestive and Kidney Diseases (NIDDK), K08DK073036.

The data reported here have been supplied by United Network for Organ Sharing as the contractor for the OPTN. The interpretation and reporting of these data are the responsibility of the authors and should in no way be seen as representing official policy of or interpretation by the OPTN or the U.S. Government.